Long Xinghua, Nephew Kenneth P
Medical Sciences, Indiana University School of Medicine, Bloomington, Indiana 47405, USA.
J Biol Chem. 2006 Apr 7;281(14):9607-15. doi: 10.1074/jbc.M510809200. Epub 2006 Feb 3.
The antiestrogen fulvestrant (ICI 182,780) causes immobilization of estrogen receptor-alpha (ERalpha) in the nuclear matrix accompanied by rapid degradation by the ubiquitin-proteasome pathway. In this study we tested the hypothesis that fulvestrant induces specific nuclear matrix protein-ERalpha interactions that mediate receptor immobilization and turnover. A glutathione S-transferase (GST)-ERalpha-activating function-2 (AF2) fusion protein was used to isolate and purify receptor-interacting proteins in cell lysates prepared from human MCF-7 breast cancer cells. After SDS-PAGE and gel excision, mass spectrometry was used to identify two major ERalpha-interacting proteins, cytokeratins 8 and 18 (CK8.CK18). We determined, using ERalpha-activating function-2 mutants, that helix 12 (H12) of ERalpha, but not its F domain, is essential for fulvestrant-induced ERalpha-CK8 and CK18 interactions. To investigate the in vivo role of H12 in fulvestrant-induced ERalpha immobilization/degradation, transient transfection assays were performed using wild type ERalpha,ERalpha with a mutated H12, and ERalpha with a deleted F domain. Of those, only the ERalpha H12 mutant was resistant to fulvestrant-induced immobilization to the nuclear matrix and protein degradation. Fulvestrant treatment caused ERalpha degradation in CK8.CK18-positive human breast cancer cells, and CK8 and CK18 depletion by small interference RNAs partially blocked fulvestrant-induced receptor degradation. Furthermore, fulvestrant-induced ERalpha degradation was not observed in CK8 or CK18-negative cancer cells, suggesting that these two intermediate filament proteins are necessary for fulvestrant-induced receptor turnover. Using an ERalpha-green fluorescent protein construct in fluorescence microscopy revealed that fulvestrant-induced cytoplasmic localization of newly synthesized receptor is mediated by its interaction with CK8 and CK18. In summary, this study provides the first direct evidence linking ERalpha immobilization and degradation to the nuclear matrix. We suggest that fulvestrant induces ERalpha to interact with CK8 and CK18, drawing the receptor into close proximity to nuclear matrix-associated proteasomes that facilitate ERalpha turnover.
抗雌激素药物氟维司群（ICI 182,780）可使雌激素受体α（ERα）固定于核基质中，并通过泛素 - 蛋白酶体途径迅速降解。在本研究中，我们验证了如下假说：氟维司群诱导特异性核基质蛋白与ERα相互作用，从而介导受体的固定及更新。利用谷胱甘肽S - 转移酶（GST）-ERα激活功能区2（AF2）融合蛋白，从人MCF - 7乳腺癌细胞制备的细胞裂解物中分离并纯化与受体相互作用的蛋白。经十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）及凝胶切胶后，采用质谱鉴定出两种主要的与ERα相互作用的蛋白，即细胞角蛋白8和18（CK8、CK18）。我们利用ERα激活功能区2突变体确定，ERα的螺旋12（H12）而非其F结构域，对于氟维司群诱导的ERα与CK8及CK18的相互作用至关重要。为研究H12在氟维司群诱导的ERα固定/降解中的体内作用，使用野生型ERα、H12突变的ERα以及F结构域缺失的ERα进行瞬时转染实验。其中，只有ERα H12突变体对氟维司群诱导的固定于核基质及蛋白降解具有抗性。氟维司群处理导致CK8、CK18阳性的人乳腺癌细胞中ERα降解，而通过小干扰RNA使CK8和CK18缺失可部分阻断氟维司群诱导的受体降解。此外，在CK8或CK18阴性的癌细胞中未观察到氟维司群诱导的ERα降解，这表明这两种中间丝蛋白对于氟维司群诱导的受体更新是必需的。利用荧光显微镜下的ERα - 绿色荧光蛋白构建体显示，氟维司群诱导的新合成受体的细胞质定位是由其与CK8和CK18的相互作用介导的。总之，本研究提供了首个将ERα固定和降解与核基质联系起来的直接证据。我们认为，氟维司群诱导ERα与CK8和CK18相互作用，使受体靠近与核基质相关的蛋白酶体，从而促进ERα的更新。
